New Approaches to Better Assess Pain in Animals and Treat Pain in People: Highlights From SfN 2017

The following is the second of a two-part report on research presented at the 2017 Society for Neuroscience annual meeting, which took place November 11-15 in Washington, DC. See Part 1 here. Also see a previous report from SfN here.

Jordan McCall, Robert Gereau, and colleagues at Washington University, St. Louis, US, in collaboration with mathematician Carl Hammarsten, Lafayette College, Easton, US, are using a novel approach to analyze animal behavior, which could be expanded to other analyses as well, such as neuronal morphology. During a poster presentation, McCall noted that current methods of animal behavioral analysis “rely on preconceived notions regarding the important observed features of the dataset, rather than unbiased assessment of the fundamental structure of the data.”

The investigators are using persistent homology to generate that unbiased assessment. Persistent homology is a mathematical method used to compute preserved movements or deformations within a given space, such as stretching, bending, or crumpling (so-called topological data). To translate this method to animal behavior, researchers choose a space within which to analyze topological data. An animal is represented by points, and the points are then each surrounded by a ball. The radii of the balls are expanded until the balls begin to touch, and when they touch they create lines (two balls touching) or triangles (three balls touching), in addition to other structures. Based on the size of the radii, the structures change, but those structures that persist for a given radii are plotted on a graph―hence, persistent homology.

A persistent homology graph plots death versus birth, with death representing loss of a persistent structure and birth representing gain of a persistent structure over time, which corresponds to inherent structure (the permanent quality of the structure). A diagonal line is placed between the death and birth axes; points that lie too close or too far from the diagonal are considered noise and are removed from analysis. Using this approach, different experimental groups within a cohort of animals can be defined.

McCall showed that these methods can be applied to animal behavior by examining locomotor behavior of more than 100 stressed or unstressed mice. Mice were either subjected to restraint (stress-induced anxiety) or not (control) and then assayed using the open field test. Persistent homological analyses revealed two distinct groups: one group comprised the restrained animals that were presumed to have anxiety, while the other group comprised the control animals.

Next, McCall tested whether the homological features from the two groups could be used to predict whether new mice were subjected to stress. Using only the previously established homological feature (the location within the open field where the mouse spends time during the open field test) of stressed versus unstressed mice, he achieved 90 percent accuracy in identifying new mice that were stressed.

Although persistent homology could accurately predict stressed versus unstressed mice, it was unable to detect reversal of stress when propranolol, an anxiolytic that reverses stress behavior in traditional analyses, was administered to stressed mice. McCall later told PRF, “We think this is because propranolol-reversal of stress-induced anxiety is not the same thing as never being stressed—something not detectable in traditional analyses—but we are doing follow-up experiments to test that hypothesis.” Although propranolol decreases stress-induced anxiety, it does not fully reduce it in mice to the same levels seen in mice that had never experienced that anxiety.

Potentially, these new analyses could also be used for pain. McCall used complete Freund's adjuvant (CFA) to induce inflammation in both plantar hindpaws of mice and then performed an open field test three weeks later, a time at which the animals do not display anxiety-like behaviors. Using the previously defined homological features of stressed and unstressed mice, 100 percent of the CFA and saline (control) mice were classified into the unstressed group. “We need to follow up with more pain-related data and maybe build a new 'training set' from mice in pain conditions,” McCall told PRF. Building a new “training set” would consist of defining the homological features that mice with various pain conditions display, similar to how McCall defined the features of stressed and unstressed mice.

Together, the data suggest that persistent homology can be used to identify important animal behavioral features in an unbiased fashion.

Yaakov Levine, SetPoint Medical Corporation, Valencia, US, presented a novel, patented implantable device for electrical stimulation of the vagus nerve. The miniature device is an integrated implantable pulse generator containing a rechargeable battery, an application-specific integrated circuit (ASIC), telemetry, as well as an electrode-containing channel that fits directly on the nerve itself. It is hoped that this design will result in easier and safer implantations. An external controller, known as a prescription pad, is used to deliver the treatment and record communication from the implant.

Levine also presented previously published results on the use of an implanted device for treatment of rheumatoid arthritis (RA; Koopman et al., 2016). RA is an autoimmune disease that results in the destruction of cartilage around joints and painful swelling that can lead to bone erosion and joint deformity. Typically, biologics that inhibit components of the immune system are used to treat RA, including antibodies that inhibit the cytokine tumor necrosis factor (TNF). However, biologics are very expensive, relieve RA symptoms in only about half of patients, and carry risks of serious side effects.

To address this limitation, Levine and colleagues turned to the inflammatory reflex, which is a cytokine-inhibiting pathway controlled by the vagus nerve. This nerve signals to the splenic nerve, which results in release of noradrenaline in the spleen. In response, certain splenic T cells then release acetylcholine, which is recognized by macrophages and results in decreased release of systemic proinflammatory factors, including TNF. Levine and colleagues hypothesized that reduction of those factors by the inflammatory reflex should lead to decreased joint inflammation, pain, and damage.

First, to test if vagus nerve stimulation (VNS) and activation of the inflammatory reflex could prevent TNF production in people, the researchers studied epilepsy patients with no history of inflammatory or autoimmune disorders. These patients were implanted with a vagus nerve-stimulating device from Cyberonics, a medical device company in Houston, US, to treat their epilepsy, and the researchers collected whole blood samples before and after device implantation. Since these patients were not inflamed, the investigators exposed the blood samples to endotoxin, which causes TNF production. Stimulation of the vagus nerve with the device significantly inhibited not only endotoxin-induced TNF production in the blood samples, but two other proinflammatory cytokines as well (IL-1β and IL-6).

Next, the researchers studied the effects of VNS in patients with active RA (those who had at least four tender and four swollen joints out of 28 total assessed joints), splitting the patients into two groups. In the first group, patients had been unresponsive to methotrexate (an anti-inflammatory drug), and had never received an anti-TNF biologic. Patients in the second, more refractory group also had not responded to methotrexate, or to two biologics with different mechanisms of action. All patients were implanted with the vagus nerve-stimulating device and received the same treatment protocol, consisting of a period of no VNS to allow for implantation recovery after surgery; a period of daily VNS with increasing intensity; a period of no VNS (therapy withdrawal); and a period where daily VNS was restarted. To study the effects of VNS on TNF production, peripheral whole blood was taken before device implantation and at the end of each period post-implantation.

After the first period of daily VNS, TNF was significantly reduced from baseline. However, TNF levels increased when VNS was stopped, but again decreased after VNS was restarted. Thus, active VNS appeared responsible for the inhibition of TNF.

The researchers also observed RA signs and symptoms by counting swollen and tender joints out of the 28 total joints, surveying patients for disease activity, and measuring serum levels of C-reactive protein (CRP). The standard score compiled from these factors is known as the DAS28-CRP score, where a high score indicates severe disease activity. Similar to TNF production, when the patients received VNS the DAS28-CRP score was decreased, and when the device was off the DAS28-CRP score was increased, in both patient groups. These data suggest that active VNS decreases not just TNF production but also RA disease severity.

At the initial cessation of treatment, the researchers divided the two patient cohorts into groups based upon American College of Rheumatology (ACR) response criteria, which are used to measure the effectiveness of RA treatments in clinical trials. ACR20 is 20 percent or greater improvement, ACR50 is 50 percent or greater improvement, and ACR70 is 70 percent or greater improvement.

In the first patient group, about 71 percent, 57 percent, and 29 percent of patients fell into ACR20, ACR50, and ACR70, respectively, with 29 percent achieving remission. In the second group, approximately 70 percent, 30 percent, and 0 percent of patients met ACR20, ACR50, and ACR70 criteria, respectively, with no patients achieving remission. Finally, the researchers found that IL-6 serum levels were elevated in VNS non-responders compared to responders.

Collectively, these results suggest that VNS can be used to inhibit systemic cytokine production and reduce RA disease severity. Long-term safety and efficacy data are expected this spring.

Ashley Cowie is a PhD candidate at the Medical College of Wisconsin, Milwaukee, US.